Site-specific incorporation of phosphoserine into proteins in Escherichia coli

Abstract

Nucleic acids encoding mutant elongation factor proteins (EF-Sep), phosphoseryl-tRNA synthetase (SepRS), and phosphoseryl-tRNA (tRNA Sep ) and methods of use in site specific incorporation of phosphoserine into a protein or polypeptide are described. Typically, SepRS preferentially aminoacylates tRNA Sep with O-phosphoserine and the tRNA Sep recognizes at least one codon such as a stop codon. Due to the negative charge of the phosphoserine, Sept-tRNA Sep does not bind elongation factor Tu (EF-Tu). However, mutant EF-Sep proteins are disclosed that bind Sep-tRNA Sep and protect Sep-tRNA Sep from deacylation. In a preferred embodiment the nucleic acids are on vectors and are expressed in cells such as bacterial cells, archeaebacterial cells, and eukaryotic cells. Proteins or polypeptides containing phosphoserine produced by the methods described herein can be used for a variety of applications such as research, antibody production, protein array manufacture and development of cell-based screens for new drug discovery.

We claim: 
     
       1. A polynucleotide encoding an elongation factor (EF-Sep) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein the amino acid sequence comprises the amino acids of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 at positions corresponding to amino acid number 67, 216, 217, 219, 229, and 274 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and
 wherein the EF-Sep can bind to a Sep-tRNA Sep  and can catalyze the covalent transfer of the phosphoserine amino acid of the Sep-tRNA Sep  onto a polypeptide. 
 
     
     
       2. The polynucleotide of  claim 1 , wherein the EF-Sep comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 
     
     
       3. The polynucleotide of  claim 2 , further comprising an expression control sequence which allows expression of the polynucleotide in a cell. 
     
     
       4. An expression vector comprising the polynucleotide of  claim 3 . 
     
     
       5. A cell comprising the polynucleotide of  claim 3 . 
     
     
       6. The cell of  claim 5 , wherein the polynucleotide is integrated into the genome of the cell. 
     
     
       7. The cell of  claim 5 , wherein the polynucleotide is integrated into an expression vector transformed or transfected into the cell. 
     
     
       8. The cell of  claim 5 , wherein the cell is an  Escherichia coli  cell. 
     
     
       9. A polynucleotide encoding an EF-Sep,
 wherein the EF-Sep comprises the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and 
 wherein the EF-Sep can bind to a Sep-tRNA Sep  and can catalyze the covalent transfer of the phosphoserine amino acid of the Sep-tRNA Sep  onto a polypeptide. 
 
     
     
       10. The polynucleotide of  claim 9 , wherein the EF-Sep comprises the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:1. 
     
     
       11. The polynucleotide of  claim 10 , wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:5 that encodes the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO: 1. 
     
     
       12. The polynucleotide of  claim 9 , wherein the EF-Sep comprises the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:2. 
     
     
       13. The polynucleotide of  claim 12 , wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:6 that encodes the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:2. 
     
     
       14. The polynucleotide of  claim 9 , wherein the EF-Sep comprises the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:3. 
     
     
       15. The polynucleotide of  claim 9 , wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:7 that encodes the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:3. 
     
     
       16. The polynucleotide of  claim 9 , wherein the EF-Sep comprises the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:4. 
     
     
       17. The polynucleotide of  claim 16 , wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:8 that encodes the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:4. 
     
     
       18. The polynucleotide of  claim 9 , wherein the EF-Sep comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 
     
     
       19. The polynucleotide of  claim 9 , further comprising an expression control sequence. 
     
     
       20. An expression vector comprising the polynucleotide of  claim 19 . 
     
     
       21. A cell comprising the polynucleotide of  claim 19 . 
     
     
       22. The cell of  claim 21 , wherein the polynucleotide is integrated into the genome of the cell. 
     
     
       23. The cell of  claim 21 , wherein the polynucleotide is integrated into an expression vector transformed or transfected into the cell. 
     
     
       24. The cell of  claim 21 , wherein the cell is an  Escherichia coli  cell. 
     
     
       25. A kit comprising
 (i) a polynucleotide encoding an elongation factor (EF-Sep); 
 (ii) an expression vector comprising a polynucleotide encoding an elongation factor (EF-Sep); or 
 (iii) a cell comprising an expression vector comprising a polynucleotide encoding an elongation factor (EF-Sep), 
 wherein the EF-Sep comprises an amino acid sequence at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein the amino acid sequence comprises the amino acids of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 at positions corresponding to amino acid number 67, 216, 217, 219, 229, and 274 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and 
 wherein the EF-Sep can bind to a Sep-tRNA Sep  and can catalyze the covalent transfer of the phosphoserine amino acid of the Sep-tRNA Sep  onto a polypeptide. 
 
     
     
       26. A kit comprising
 (i) a polynucleotide encoding an elongation factor (EF-Sep); 
 (ii) an expression vector comprising a polynucleotide encoding an elongation factor (EF-Sep); or 
 (iii) a cell comprising an expression vector comprising a polynucleotide encoding an elongation factor (EF-Sep), 
 wherein the EF-Sep comprises the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and 
 wherein the EF-Sep can bind to a Sep-tRNA Sep  and can catalyze the covalent transfer of the phosphoserine amino acid of the Sep-tRNA Sep  onto a polypeptide.